Virus dissemination and target organs.

<p>Following inoculation with CHIKV through a mosquito bite, the virus directly enters the subcutaneous capillaries, with some viruses infecting susceptible cells in the skin, such as macrophages or fibroblasts and endothelial cells. Local viral replication seems to be minor and limited in time, with the locally produced virus probably being transported to secondary lymphoid organs close to the site of inoculation. The blood carries most viruses, as free virions or in the form of infected monocytes, to the target organs, the liver, muscle, joints, and remote lymphoid organs. In these tissues, infection is associated with a marked infiltration of mononuclear cells, including macrophages, particularly when viral replication occurs. The pathological events associated with tissue infection are mostly subclinical in the liver (hepatocyte apoptosis) and lymphoid organs (adenopathy), whereas mononuclear cell infiltration and viral replication in the muscles and joints are associated with very strong pain, with some of the patients presenting arthritis. * Guillain-Barré syndrome and encephalitis are very rare events. † True arthritis remains a rare event (from 2% to 10%); see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001446#pntd-0001446-t002" target="_blank">Table 2</a>.</p

The macrophage is central to chronic signs of chikungunya disease.

<p>Macrophage infiltration, under the control of MCP-1/CCL-2, is a critical feature of damaged tissues. The inflammatory effectors IL-6, IL-8, MCP-1/CCL-2, MMP2, and INF-α are specifically expressed in the tissues of patients with chronic chikungunya, who have high IFN-α and IL-12 mRNA levels in their circulating leukocytes. This classical inflammatory process may be regulated by HGF and eotaxin, which have different expression profiles during the recovery phase in patients with chikungunya, depending on whether or not these patients go on to develop chronic disease. HGF also promotes muscle regeneration. Once they have infiltrated the joint or muscle, the macrophages are activated and regulate the local Th1/Th2 balance as a function of their own activation status (classical/M1 or alternative/M2). GM-CSF and HGF, which have M1 and M2 effector activities, respectively, may modulate this balance as they are differentially expressed in acute and chronic chikungunya. CHIKV persists in infected macrophages only in patients with a chronic rheumatic syndrome. The reciprocal influences connecting viral persistence and local inflammation are not known. Solid arrows: activation. Solid stopped lines: regulation. Dotted arrows: expression.</p

Acute chikungunya symptoms in typical and atypical forms in adults, children, and newborns.

a<p>Atypical forms were noticed but their prevalence was not evaluated.</p>b<p>Retro-orbital pain.</p

Mechanisms of CHIKV persistence and tissue inflammation in patients with chronic disease.

<p>(1) Months after the acute infection, monocytes, T cells, and natural killer (NK) cells are still attracted to the inflamed joint, where they become activated. (2) The infection of macrophages in joints is associated with local inflammation and the production of cytokines, chemokines, and pro-inflammatory effectors, such as MCP-1/CCL-2, IL-8, IL-6, IFN-α, and MMP2. (3) The phagocytosis of apoptotic bodies from infected cells probably contributes to viral persistence. Nevertheless, the beneficial or deleterious effect of local inflammation on viral persistence remains unclear. (4) When it occurs, arthritis is accompanied by high rates of fibroblast apoptosis and cartilage destruction. Chronic inflammation probably plays a major role in this damage and associated pain. (5) The potential relationship between local inflammation of the joint and a state of systemic activation, as demonstrated by the presence of inflammation markers in plasma and blood cells, remains unclear.</p

Chronic chikungunya symptoms.

*<p><i>p</i><0.01 Khi-2 between infected time 0 and chronic at 12 months (calculated by us).</p>a<p>Low ALT/ASAT level related to persistence (<i>p</i><0.01) like platelet count.</p>b<p>Lower level of creatinine <i>p</i> = 0.036.</p>c<p>No difference chronic versus recovered.</p>d<p>American College of Rheumatology criteria for rheumatoid arthritis in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001446#pntd.0001446-Chopra2" target="_blank">[28]</a>; note that tenosynovitis is prominent (as seen by Hoarau et al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001446#pntd.0001446-Hoarau1" target="_blank">[41]</a>).</p><p>IA, inflammatory arthritis; IE, inflammatory, erosive; MRI, magnetic resonance imaging; ND, not detected or prevalence not evaluated; NR, not reported; NSA, non-specific arthritis.</p

Anti-CHIKV antibodies demonstrate polymorphic epitope recognition against different CHIKV isolates.

<p><i>A,</i> Schematic representation of the coding regions of CHIKV genome. <i>B,</i> Total cell lysates were prepared from CHIKV-infected 293T cells and mock-infected 293T cells. Lysates were subjected to SDS-PAGE gel electrophoresis (Left lane – mock-infected lysates, right lane – CHIKV-infected lysates) and probed with sera from CHIKV-infected macaques at a dilution of 1∶2,000, followed by HRP-conjugated anti-monkey IgG secondary antibodies. The arrows at the top panel indicate the CHIKV antigens (Capsid and E2) detected by antibodies in the sera of CHIKV-infected macaques. Arrows at the middle panel (non-structural proteins) and the lower panel (structural proteins) indicate CHIKV proteins detected by corresponding CHIKV antigen-specific rabbit sera performed at a dilution of 1∶2,000 followed by HRP-conjugated anti-rabbit IgG secondary antibodies. Housekeeping protein GAPDH was probed with a specific anti-GAPDH antibody (Biolegend) as an indicator for loading control. Sizes of molecular weight markers are indicated in the left part of the diagram. <i>C,</i> Purified CHIKV virions (IMT and SGP11) were prepared under reducing (100°C, 5 min+DTT) or non-reducing conditions and subjected to SDS-PAGE gel electrophoresis, then probed with sera from CHIKV-infected macaques at a dilution of 1∶2,000, followed by secondary HRP-conjugated anti-monkey IgG. Sizes of molecular weight markers are indicated on the left part of the diagram. Non-annotated arrows represent the presence of disulfide bonds dependent conformational epitopes recognized by antibodies from the sera of CHIKV-infected macaques.</p

Unique Epitopes Recognized by Antibodies Induced in Chikungunya Virus-Infected Non-Human Primates: Implications for the Study of Immunopathology and Vaccine Development

<div><p>Chikungunya virus (CHIKV) is an <i>Alphavirus</i> that causes chronic and incapacitating arthralgia in humans. Although patient cohort studies have shown the production of CHIKV specific antibodies, the fine specificity of the antibody response against CHIKV is not completely defined. The macaque model of CHIKV infection was established due to limitations of clinical specimens. More importantly, its close relation to humans will allow the study of chronic infection and further identify important CHIKV targets. In this study, serum samples from CHIKV-infected macaques collected at different time-points post infection were used to characterize the antibody production pattern and kinetics. Results revealed that anti-CHIKV antibodies were neutralizing and the E2 glycoprotein, Capsid, nsP1, nsP3 and nsP4 proteins were targets of the anti-CHIKV antibody response in macaques. Furthermore, linear B-cell epitopes recognized by these anti-CHIKV antibodies were identified, and mapped to their structural localization. This characterizes the specificity of anti-CHIKV antibody response in macaques and further demonstrates the importance of the different regions in CHIKV-encoded proteins in the adaptive immune response. Information from this study provides critical knowledge that will aid in the understanding of CHIKV infection and immunity, vaccine design, and pre-clinical efficacy studies.</p></div

Mapping of CHIKV B-cell epitopes within CHIKV proteome.

<p>Sera from CHIKV-infected macaques (16 and 180 dpi) were diluted 1∶2,000 and subjected to peptide-based ELISA with a peptide library covering the CHIKV proteome, using pooled peptides (named P1, P2, etc) from the structural (<i>A,</i> Capsid, <i>B,</i> E2) and non-structural (<i>C,</i> nsP1, <i>D,</i> nsP3 and <i>E,</i> nsP4) proteins. Sera from non-infected macaques were used as negative controls. *Pooled peptides were considered to contain positive linear B-cell epitopes when OD values obtained with sera from CHIKV-infected macaques were above mean +3 SD of the OD values obtained with sera from non-infected macaques. Data are presented as OD values obtained using sera from infected macaques, minus the OD values obtained using sera from non-infected macaques, for the corresponding pooled peptides. Data represent an average of two independent experiments (mean ± SD).</p

Analysis of anti-CHIKV antibodies recognizing linear B-cell epitopes.

<p><i>A,</i> Serum samples from CHIKV-infected macaques (n = 1–3) were collected at 9, 16, 100 and 180 dpi. Peptide-based ELISA covering the CHIKV proteome, encompassing the non-structural and structural proteins, was performed with sera diluted at 1∶2,000. Regions of amino acid sequence corresponding to the identified linear B-cell epitopes are indicated on the genome organization schematic diagram. <i>B,</i> The percentage of antibody recognition for various CHIKV epitopes is indicated on the pie-charts. The percentage was calculated according to the equation: % antibody recognition = 100 x (OD values from pooled peptide group/sum of OD values from all pooled peptide groups).</p

Localization of linear CHIKV B-cell epitopes within the CHIKV proteome.

<p><i>A,</i> Schematic representation of identified B-cell epitopes in nsP1, nsP3, nsP4 and Capsid protein. <i>B,</i> Schematic representation of identified B-cell epitopes in E2 glycoprotein. Epitopes in the E2 glycoprotein were located based on structural data retrieved from PDB number: 3N44. Epitopes in the Capsid, nsP1, nsP3 and nsP4 proteins were located based on structures predicted by I-TASSER server.</p